In this four year two phase (R21/R33) project we will integrate and apply new approaches for obtaining broad systems level views of protein expression in cancer research. The overall approach will advance the study of proteomes by more rapidly identifying proteins, precisely measuring the relative abundances for all detected proteins, and providing much greater sensitivity than existing methodologies. Our approach will utilize proteome-wide stable isotope and biotin labeling of cysteine-containing polypeptides combined with new approaches that use ultra-high sensitivity Fourier transform ion cyclotron resonance mass spectrometry. The approach will be at least 2 to 3 orders of magnitude more sensitive than existing 2-D PAGE methodologies and able to rapidly identify and measure relative expression levels for thousands of proteins in a single analysis. Phase 1 of this project will integrate and provide an initial demonstration of methods that include the sample processing for mouse B16 melanoma cells from culture, validate the use of new accurate mass tag and multiplexed-MS/MS methods for protein identification, and demonstrate the precise determination of relative protein abundances for all detected proteins from B16 and B16BL6 cell populations. Phase 2 will involve the pilot application of the technology to the study of proteome changes that occur as cells progress from low or nonmetastatic states, to a highly invasive and metastatic phenotype, using the B16 melanoma system as a model. The technology to be applied will enable ultra-sensitive (attomole level, and anticipated to be better) proteome-wide precise profiling of proteins from cells maintained in culture and from tissues (obtained by micro-dissection). The results will provide an abundance of new information on protein expression, and enable precise measurements of differences in relative protein expression levels as a function of cell type, developmental stage, metastasis, etc. A product of this research will be the first application of a high throughput technology for obtaining precise proteome displays that may be expected to illuminate the complex mechanisms and pathways relevant to cancer.